Preparation method of composite mold inhibitor and composite mold inhibitor

Abstract

The application relates to the technical field of mold removers, in particular to a preparation method of a composite mold remover and the composite mold remover. The preparation method of the composite mold remover comprises the following steps: S1. preparing modified montmorillonite; S2. culturing Brevibacillus parvus in a culture medium, obtaining Brevibacillus parvus bacterial liquid after the culturing is completed, and then preparing the obtained bacterial liquid into dry powder; S3. mixing methyl mercaptoacetate, dimethyl trisulfide and 3-methyl butyl sulfide S-methyl ester with water, and then adding the dry powder to obtain a mixed solution; S4. mixing the mixed solution with the modified montmorillonite to obtain a composite mold remover preparation; and S5. drying and packaging the composite mold remover. The composite mold remover obtained by the application is applied to moldy feed, and excellent mold removal effect can be achieved after 24 hours of mold removal treatment.

Description

Technical Field

[0001] This application relates to the field of mycotoxin binders, and in particular to a method for preparing a composite mycotoxin binder and the composite mycotoxin binder. Background Technology

[0002] Mycotoxins mainly refer to the toxic metabolites produced by molds in contaminated grains. The effects of mycotoxins on grains mainly include the following four aspects.

[0003] Impact on grain quality: Mycotoxin contamination can cause grain to deteriorate, reduce its edible value, and cause economic losses; mycotoxin contamination may lead to mold, discoloration, off-odors, and other phenomena in grains, seriously affecting their appearance and taste.

[0004] Impact on human health: Ingestion of large doses of mycotoxins may cause acute complications such as liver parenchymal cell necrosis, bile duct epithelial cell hyperplasia, hepatic fatty infiltration, and liver hemorrhage. Early symptoms include fever, vomiting, loss of appetite, and jaundice, followed by ascites, lower limb edema, and rapid death. Long-term consumption of grains containing low doses of mycotoxins may have chronic health hazards, such as increasing the risk of cancer and affecting the function of the immune system.

[0005] Impact on livestock and poultry farming: Grains contaminated with mycotoxins, when used as feed, will reduce the feed intake and feed utilization rate of livestock and poultry, leading to slow growth and weight loss. Mycotoxins may also damage the reproductive system of livestock and poultry, resulting in poor reproductive performance, such as sows returning to estrus, sows being anestrus, sows not coming into estrus, sows having fewer piglets, sows having abortions, sows having stillbirths, boars having low libido, boars having low sperm motility, and boars having poor semen quality. Mycotoxins can also remain in livestock and poultry products, posing a great threat to human health and safety.

[0006] Impact on the agricultural economy: Grain losses and quality decline caused by mycotoxin contamination severely affect agricultural economic development. Simultaneously, diseases and deaths caused by mycotoxin-contaminated feed in livestock farming also result in significant economic losses for the livestock industry.

[0007] According to statistics, there are more than 300 known mycotoxins, among which aflatoxin, zearalenone, T-2 toxin, ochratoxin, vomitoxin, and fumonisin are highly toxic, widely distributed, and produce high levels of toxins.

[0008] Methods for removing mycotoxins include chemical degradation, physical degradation, and biological degradation. Chemical degradation primarily uses oxidants or strong alkalis to degrade toxins. Physical degradation methods include adsorbent washing, adsorption, solvent extraction, heat detoxification, ultraviolet detoxification, and embryo removal detoxification, with adsorbent adsorption being the most common. However, these two methods suffer from unstable and incomplete degradation effects, and can lead to the loss of nutrients and trace elements, as well as poor palatability. Biological degradation utilizes microorganisms or their degradation products to degrade toxins. It has advantages such as minimal impact on the sensory properties, palatability, and nutrients of the raw materials, and is also safe, environmentally friendly, and highly efficient.

[0009] Chinese patent application number 202310154956.6 discloses a biological mycotoxin binder for degrading mycotoxins in feed. When applied to silage that had begun to mold, the content of various toxins was measured after one week. The results showed a significant reduction in the proportion of moldy feed. Specifically, the degradation efficiency for zearalenone reached 89%, and the degradation efficiency for vomitoxin reached 90%. Aflatoxin B1 was no longer detected. The applicant argues that existing methods for microbial degradation of mycotoxins have relatively long degradation times and that the degradation rate needs further improvement. Summary of the Invention

[0010] In order to improve the degradation rate of mycotoxins and shorten the degradation time, this application provides a method for preparing a composite mycotoxin binder and the composite mycotoxin binder.

[0011] In a first aspect, this application provides a method for preparing a composite mycotoxin remover, which adopts the following technical solution.

[0012] A method for preparing a compound mycotoxin remover includes the following steps:

[0013] S1. Preparation of modified montmorillonite:

[0014] 1) Dissolve copper sulfate in water to prepare a copper sulfate solution;

[0015] 2) Add montmorillonite to the copper sulfate solution, and then add a pH adjuster to adjust the pH of the solution to 10;

[0016] 3) Add formaldehyde solution as a reducing agent and start the chemical deposition reaction at 70-75℃ for 12-14 hours;

[0017] 4) After the reaction is complete, filter, wash and dry to remove unreacted copper ions and reducing agent residues to obtain modified montmorillonite;

[0018] S2. Bacillus laterosporus is cultured in a culture medium to obtain Bacillus laterosporus bacterial solution after the culture is completed. Then the obtained bacterial solution is made into dry powder.

[0019] S3. Mix methyl mercaptoacetate, dimethyl trisulfide, 3-methylbutyrate (S-methyl ester) with water, then add the dry powder to obtain a mixture;

[0020] S4. Mix the mixture with modified montmorillonite to obtain a composite mycotoxin binder formulation;

[0021] S5. After drying the compound mycotoxin remover, package it.

[0022] By employing the above-mentioned technical solutions, *Bacillus laterosporus* can produce antimicrobial peptides and polyketides, which have inhibitory effects on various molds. Antimicrobial peptides can bind to lipopolysaccharide / lipid II molecules on the mold cell membrane, altering cell membrane permeability and thus inhibiting mold growth. Polyketides inhibit mold reproduction by affecting relevant gene pathways in the mold cell membrane, such as ATP synthesis, peptidoglycan biosynthesis, membrane transport, and cell metabolism. *Bacillus laterosporus* can secrete chitinase, which degrades chitin in the fungal cell wall, disrupting the structural integrity of the mold and thus inhibiting its growth. During its growth, *Bacillus laterosporus* consumes nutrients in the environment, competing with molds for limited nutrient resources, thereby limiting mold growth and reproduction.

[0023] Methyl mercaptoacetate (MGA) can disrupt the cell membranes of microorganisms, causing leakage of cell contents and thus inhibiting or killing bacteria. The sulfhydryl group can react with sulfhydryl enzymes in the enzyme system of microorganisms, interfering with their normal metabolism and thus inhibiting microbial growth. Similarly, MGA can disrupt the cell membranes of fungi, affecting cell wall synthesis and leading to fungal cell death. Furthermore, it can interfere with fungal metabolic processes, inhibiting spore formation and germination, thereby achieving an antifungal effect.

[0024] Dimethyl trisulfide can directly harm fungi by disrupting their cell structure, affecting their normal growth and reproduction, thus achieving an anti-mold effect. It can penetrate mold cells, damaging their proteins and protoplasmic membranes, leading to water loss and making the mold cells unable to survive. Simultaneously, it can prevent the leakage of various ions, enzymes, coenzymes, and intermediate products from within the mold cells, forming a virtuous cycle and enhancing the anti-mold effect. It can also react with the chromosomal material of mold cells, inhibiting chromosome division or causing mutations, affecting the division, growth, and morphology of mold cells, thereby achieving the purpose of mold prevention.

[0025] 3-Methylbutyrate (S-methyl ester) can disrupt the cell membranes of microorganisms, causing leakage of cell contents and thus inhibiting or killing bacteria. Its ester and sulfur groups enable it to react with the enzyme systems within microorganisms, interfering with their normal metabolism and inhibiting their growth. It can also disrupt the cell membranes of fungi, affecting cell wall synthesis and leading to fungal cell death. Furthermore, it can interfere with fungal metabolic processes, inhibiting spore formation and germination, thereby achieving an antifungal effect.

[0026] By grafting copper ions onto montmorillonite using chemical deposition, the adsorption effect of montmorillonite on molds is improved. Furthermore, copper ions can enhance the removal efficiency of Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester of molds. Modified montmorillonite exhibits strong adsorption properties, effectively absorbing molds from feed. It can also adsorb Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester, resulting in a more precise inhibitory effect from these substances, leading to rapid mold killing. Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and S-methyl 3-methylbutyrate ester work synergistically. Methyl mercaptoacetate, dimethyl trisulfide, and S-methyl 3-methylbutyrate ester can rapidly eliminate mycotoxins, while simultaneously inhibiting the release of substances from Bacillus laterosporus, resulting in a slower release of mycotoxin-reducing substances and prolonging the effective action time of the mycotoxin-reducing agent, thus allowing for more thorough removal of mold.

[0027] Furthermore, the concentration of the copper sulfate solution is 16-20 g / L; the weight ratio of copper sulfate to montmorillonite is 1:(5-12); and the weight ratio of formaldehyde to copper sulfate is (3-4):5.

[0028] Furthermore, the weight ratio of copper sulfate to montmorillonite is 1:(6-8).

[0029] Furthermore, the modified montmorillonite is grafted with hexadecyltrimethylammonium bromide, and the grafting method is as follows:

[0030] a) Dissolve hexadecyltrimethylammonium bromide in water to obtain a hexadecyltrimethylammonium bromide solution, and add methyl p-toluenesulfonate to the hexadecyltrimethylammonium bromide solution to obtain a treatment solution;

[0031] b) Add the modified montmorillonite to the treatment solution, mix, and then reflux at 70-85℃ for 2-4 hours;

[0032] c) After the reaction is complete, the slurry is ultrasonically dispersed, the supernatant is removed by centrifugation, the precipitate is collected and washed; the washed precipitate is dried at 80-90℃ to obtain hexadecyltrimethylammonium bromide-modified montmorillonite.

[0033] By adopting the above technical solution, grafting hexadecyltrimethylammonium bromide increases the interlayer distance of montmorillonite, which on the one hand further improves the adsorption effect of montmorillonite, and on the other hand allows water molecules to enter the interlayer of montmorillonite better, thereby improving the water solubility of montmorillonite.

[0034] Furthermore, the weight ratio of hexadecyltrimethyl bromide to methyl p-toluenesulfonate is 1:(0.1-0.3); the weight ratio of hexadecyltrimethyl bromide to modified montmorillonite is 1:(35-42).

[0035] Furthermore, in S3, methyl mercaptoacetate, dimethyl trisulfide, 3-methylbutyrate S-methyl ester, 3-mercaptopropyltrimethoxysilane are mixed with water, and then dry powder is added to obtain a mixture.

[0036] By adopting the above technical solution, 3-mercaptopropyltrimethoxysilane containing thiol groups is physically mixed with montmorillonite, thereby enhancing the colonization effect of montmorillonite on probiotics through physical adsorption.

[0037] Secondly, this application provides a compound mycotoxin remover, using the following technical solution.

[0038] A compound mycotoxin remover comprises the following raw materials in parts by weight: 5-15 parts of Bacillus retroflexus, 15-25 parts of methyl mercaptoacetate, 10-20 parts of dimethyl trisulfide, 15-35 parts of 3-methylbutyrate S-methyl ester, 15-30 parts of modified montmorillonite, 5-9 parts of 3-mercaptopropyltrimethoxysilane, and 20-40 parts of water.

[0039] Furthermore, the weight ratio of the methyl mercaptoacetate, dimethyl trisulfide, 3-methylbutyrate S-methyl ester to Bacillus lateralis is (8-12):1.

[0040] Furthermore, when applied to moldy feed, the weight ratio of moldy grain raw materials or feed to compound mycotoxin binder is (400-500):1.

[0041] By adopting the above technical solution, the compound mycotoxin binder can be applied to moldy feed, such as corn flour, and a relatively small amount of compound mycotoxin binder can achieve a high mycotoxin removal efficiency.

[0042] In summary, this application has the following beneficial effects:

[0043] This application describes a compound mycotoxin remover prepared by combining modified montmorillonite with Bacillus lateralis, methyl mercaptoacetate, dimethyl trisulfide, and S-methyl 3-methylbutyrate. When applied to moldy corn flour, after 24 hours of mycotoxin removal treatment, the removal rate of zearalenone from moldy corn flour reached 96.4-99.8%, the removal rate of aflatoxin B1 from moldy corn flour reached 96.6-99.9%, and the removal rate of vomitoxin from moldy corn flour reached 96.1-99.5%. Detailed Implementation

[0044] The present application will be further described in detail below with reference to the embodiments.

[0045] Example of raw material and intermediate preparation

[0046] raw material

[0047] All raw materials used in the embodiments of this application are commercially available.

[0048] Bacillus laterosporus is a commercially available product.

[0049] Copper sulfate, CuSO4·5H2O, analytical grade;

[0050] pH adjuster, 40% sodium hydroxide solution;

[0051] Hexadecyltrimethylammonium bromide, analytical grade;

[0052] Methyl p-toluenesulfonate, analytical grade;

[0053] Methyl mercaptoacetate, analytical grade;

[0054] Dimethyl trisulfide, analytical grade;

[0055] 3-Methylbutyrate S-methyl ester, analytical grade;

[0056] Modified montmorillonite, analytical grade;

[0057] 3-Mercaptopropyltrimethoxysilane, analytical grade.

[0058] Preparation Example

[0059] Preparation Example 1

[0060] A modified montmorillonite, the preparation method of which is as follows:

[0061] 1) Dissolve 200g of copper sulfate in water to prepare a copper sulfate solution with a concentration of 18g / L;

[0062] 2) Add 1400g of montmorillonite to the copper sulfate solution obtained in step 1), and then add a pH adjuster to adjust the pH of the solution to 10; the weight ratio of copper sulfate to montmorillonite is 1:7;

[0063] 3) Add 20L of formaldehyde solution with a concentration of 6g / L as a reducing agent, and start the chemical deposition reaction at 70℃ for 12 hours;

[0064] 4) After the reaction is complete, filter, wash and dry to remove unreacted copper ions and reducing agent residues to obtain modified montmorillonite.

[0065] Preparation Example 2

[0066] Unlike Preparation Example 1, in Preparation Example 2, step 2) uses 1200g of montmorillonite and the weight ratio of copper sulfate to montmorillonite is 1:6.

[0067] Preparation Example 3

[0068] Unlike Preparation Example 1, in Preparation Example 3, step 2) uses 1600g of montmorillonite and the weight ratio of copper sulfate to montmorillonite is 1:8.

[0069] Preparation Example 4

[0070] Unlike Preparation Example 1, in Preparation Example 3, step 2) uses 1000g of montmorillonite and the weight ratio of copper sulfate to montmorillonite is 1:5.

[0071] Preparation Example 5

[0072] A cetyltrimethylammonium bromide-modified montmorillonite, the preparation method of which is as follows:

[0073] a) Dissolve 100g of hexadecyltrimethylammonium bromide in 1000g of water to obtain a hexadecyltrimethylammonium bromide solution, and add 10g of methyl p-toluenesulfonate to the hexadecyltrimethylammonium bromide solution to obtain a treatment solution;

[0074] b) Add 3500g of modified montmorillonite to the treatment solution, mix, and then reflux at 80℃ for 3h.

[0075] c) After the reaction is complete, the slurry is ultrasonically dispersed, the supernatant is removed by centrifugation, the precipitate is collected, and the precipitate is washed three times with distilled water and anhydrous ethanol in turn. The washed precipitate is dried at 85°C to obtain hexadecyltrimethylammonium bromide-modified montmorillonite.

[0076] Preparation Example 6

[0077] Unlike Preparation Example 5, in Preparation Example 6b), 4200g of modified montmorillonite was added to the treatment solution.

[0078] Preparation Example 7

[0079] Unlike Preparation Example 5, in Preparation Example 7b), 3200g of modified montmorillonite was added to the treatment solution.

[0080] Preparation Example 8

[0081] Unlike Preparation Example 5, in Preparation Example 8, a) 30g of methyl p-toluenesulfonate was added to a hexadecyltrimethylammonium bromide solution.

[0082] Preparation Example 9

[0083] Unlike Preparation Example 5, in Preparation Example 9 a) 40g of methyl p-toluenesulfonate was added to a hexadecyltrimethylammonium bromide solution.

[0084] Example

[0085] Examples 1-8

[0086] A compound mycotoxin remover, the preparation method of which is as follows:

[0087] S1. Prepare modified montmorillonite according to the method in Preparation Example 1;

[0088] S2. Bacillus laterosporus was cultured in a culture medium according to the proportions in Table 1. After the culture was completed, Bacillus laterosporus was obtained as a bacterial suspension. The bacterial suspension was then made into a dry powder.

[0089] The culture medium for Bacillus laterosporus is: molasses 12-16 g / L, beef meal 6-10 g / L, magnesium sulfate 0.6-1 g / L, KH2PO4 0.3-0.7 g / L, K2HPO4 1-3 g / L, pH 7;

[0090] The cultivation conditions were: temperature 35℃, shaker speed 250r / min, inoculum size 8%, and cultivation time 30h.

[0091] S3. Mix methyl mercaptoacetate, dimethyl trisulfide, 3-methylbutyrate S-methyl ester, 3-mercaptopropyltrimethoxysilane and water according to the proportions in Table 1, and then add the dry powder to obtain a mixture.

[0092] S4. Mix the mixture with modified montmorillonite to obtain a composite mycotoxin binder formulation;

[0093] S5. After drying the compound mycotoxin remover, package it.

[0094] Table 1. Raw material ratios for Examples 1-8 (kg)

[0095] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Lateral spores of Bacillus brevis 5 10 15 10 10 10 10 10 Methyl mercaptoacetate 25 20 10 20 20 20 20 20 Dimethyl trisulfide 10 15 20 12 18 15 15 15 3-Methylbutyrate S-methyl ester 35 25 15 30 20 25 25 25 Modified montmorillonite 15 25 30 25 25 25 25 25 water 20 30 40 30 30 30 30 30 3-Mercaptopropyltrimethoxysilane 0 0 0 0 0 5 9 12

[0096] The modified montmorillonite was derived from Preparation Example 1.

[0097] Example 9

[0098] Unlike Example 2, the amount of Bacillus lateralis used in Example 9 was 7.5 kg.

[0099] Example 10

[0100] Unlike Example 2, the amount of Bacillus lateralis used in Example 10 was 5 kg.

[0101] Examples 11-13

[0102] Unlike Example 7, the modified montmorillonite in Examples 11-13 were derived from Preparation Examples 2-4, respectively.

[0103] Examples 14-18

[0104] Unlike Example 7, in Examples 14-18, the modified montmorillonite was replaced with an equal amount of hexadecyltrimethylammonium bromide-modified montmorillonite from Preparation Examples 5-9.

[0105] Comparative Example

[0106] Comparative Example 1

[0107] Unlike Example 1, in Comparative Example 1, the modified montmorillonite was replaced with an equal amount of montmorillonite.

[0108] Comparative Example 2

[0109] Unlike Example 3, in Comparative Example 2, 5 kg each of methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester were used to replace 15 kg of Bacillus lateralis.

[0110] Comparative Example 3

[0111] Unlike Example 1, in Comparative Example 3, an equal amount of methyl mercaptoacetate was used to replace dimethyl trisulfide.

[0112] Comparative Example 4

[0113] Unlike Example 1, in Comparative Example 4, an equal amount of dimethyl trisulfide was used to replace 3-methylbutyrate S-methyl ester.

[0114] Performance testing

[0115] I. Mycotoxin removal performance test

[0116] 7g of the compound mycotoxin binder prepared in each example and comparative proportion was added to 1kg of contaminated corn flour (containing 50ppm zearalenone, 50ppm aflatoxin B1, and 50ppm vomitoxin; the pH of the corn flour extract was 6). 1kg of distilled water was added to each experimental group, with three replicates per group. After thorough mixing, the mixture was treated at 30℃ for 24h. Samples from each experimental group were then accurately weighed, and the contents of zearalenone, aflatoxin B1, and vomitoxin in each sample were determined. The removal rate (%) was calculated using the following formula. The results are shown in Table 2.

[0117] Removal rate (%) = (mass of mycotoxins in the sample before treatment - mass of mycotoxins in the sample after treatment) / mass of mycotoxins in the sample before treatment × 100%.

[0118] Table 2 Performance Test Results / %

[0119] Zearalenone removal rate Aflatoxin B1 removal rate Vomiting toxin removal rate Example 1 96.6 96.1 96.4 Example 2 97.4 96.7 97.2 Example 3 96.4 95.9 96.2 Example 4 97.1 96.4 96.9 Example 5 96.9 96.2 96.7 Example 6 98.1 97.6 97.9 Example 7 98.4 97.9 98.2 Example 8 97.7 97.2 97.4 Example 9 98.2 97.5 97.8 Example 10 98.3 97.8 98.1 Example 11 98.4 97.5 97.8 Example 12 97.7 97.2 97.5 Example 13 97.1 96.6 96.9 Example 14 99.5 99.5 99.6 Example 15 99.2 99.2 99.3 Example 16 99.0 98.5 98.8 Example 17 99.2 99.0 99.1 Example 18 98.8 98.3 98.6 Comparative Example 1 83.8 84.2 82.6 Comparative Example 2 76.8 77.3 75.1 Comparative Example 3 87.8 88.6 86.8 Comparative Example 4 88.3 87.8 86.1

[0120] Based on Examples 1-18 and Comparative Examples 1-4, and in conjunction with Table 2, it can be seen that the composite mycotoxin binders obtained in Examples 1-18 have higher removal rates of zearalenone, aflatoxin B1, and vomitoxin in corn flour than those in Comparative Examples 1-4. This indicates that the composite mycotoxin binder obtained in this application has a better mycotoxin removal effect on corn flour. This may be because the modified montmorillonite in the composite mycotoxin remover of this application adsorbs zearalenone, aflatoxin B1, and vomitoxin on corn flour, while also adsorbing some Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester. This allows Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester to make accurate contact with zearalenone, aflatoxin B1, and vomitoxin. Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester then synergistically compete with each other for the survival nutrients and living space of zearalenone, aflatoxin B1, and vomitoxin, inhibiting their growth and degrading them, thus achieving rapid and efficient mycotoxin removal.

[0121] Combining Example 1 and Comparative Example 1, and referring to Table 2, it can be seen that the composite mycotoxin remover obtained in Example 1 has a higher removal rate of zearalenone, aflatoxin B1, and vomitoxin from corn flour than that in Comparative Example 1. This indicates that the combined use of modified montmorillonite with Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester helps to improve the removal efficiency of zearalenone, aflatoxin B1, and vomitoxin. This may be because after copper ions are grafted onto montmorillonite, on the one hand, the adsorption effect of montmorillonite on mold is improved, and on the other hand, copper ions can also promote the removal efficiency of mold by Bacillus laterosporus, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester.

[0122] Combining Example 3 with Comparative Examples 2-4, and referring to Table 2, it can be seen that the composite mycotoxin binder obtained in Example 3 has a higher removal rate of zearalenone, aflatoxin B1, and vomitoxin in corn flour than that in Comparative Examples 2-4. This indicates that combining *Bacillus laterosporus* with methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester can further improve the removal effect on molds. This may be because *Bacillus laterosporus*, methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester work synergistically. Methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester can quickly eliminate mycotoxins, while methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester have a certain inhibitory effect on the release of substances from *Bacillus laterosporus*, causing the mycotoxin-binding substances of *Bacillus laterosporus* to be released slowly, prolonging the effective action time of the mycotoxin binder, and allowing the mold to be removed more thoroughly.

[0123] Combining Examples 2 and 6-8, and referring to Table 2, it can be seen that the composite mycotoxin binders obtained in Examples 6-8 showed higher removal rates of zearalenone, aflatoxin B1, and vomitoxin from corn flour than those in Example 2. This indicates that the addition of 3-mercaptopropyltrimethoxysilane can improve the mycotoxin binder effect. This may be because 3-mercaptopropyltrimethoxysilane physically mixes with montmorillonite, enhancing the colonization effect of montmorillonite on probiotics through physical adsorption, thereby improving the mycotoxin binder effect. The amount of 3-mercaptopropyltrimethoxysilane added in Examples 6-7 is even more optimal.

[0124] Combining Examples 7 and 14-18, and referring to Table 2, it can be seen that the composite mycotoxin removers obtained in Examples 14-18 have higher removal rates of zearalenone, aflatoxin B1, and vomitoxin in corn flour than those in Example 7. This indicates that grafting hexadecyltrimethylammonium bromide onto modified montmorillonite can further improve the mycotoxin removal effect. This may be because grafting hexadecyltrimethylammonium bromide increases the interlayer distance of montmorillonite, which on the one hand further improves the adsorption effect of montmorillonite, and on the other hand allows water molecules to better enter the interlayer of montmorillonite, improving the water solubility of montmorillonite, thereby improving its mycotoxin removal effect.

[0125] II. Toxicity Testing

[0126] Experimental animals: 70 clean-grade ICR Kunming mice aged 6-8 weeks (weight: 18±2g, half male and half female) were fasted for 12 hours but allowed free access to water and were randomly divided into control group and experimental group.

[0127] The control group was fed rat food that was tested and found to be free of mold contamination, at a rate of 5g per day for 15 consecutive days.

[0128] The experimental group was fed rat food treated with the mycotoxin binder described in Example 7. The treatment method was as follows: the same rat food as the control group was ground into powder, treated with a mycotoxin binder at a weight ratio of 300:1, dried and pressed into rat food, and fed 5g per day for 15 consecutive days.

[0129] Experimental results:

[0130] One day after feeding: The mice in the control group and the experimental group had normal diet, increased weight, moved freely, had smooth fur, normal urination and defecation, no abnormal secretions, and no abnormal blood biochemical indicators.

[0131] Five days after feeding: The mice in both the control and experimental groups had normal diets, increased weight, moved freely, had smooth fur, normal urination and defecation, no abnormal secretions, and no abnormal blood biochemical indicators.

[0132] After 10 days of feeding: The mice in the control group and the experimental group had normal diet, increased weight, moved freely, had smooth fur, normal urination and defecation, no abnormal secretions, and no abnormal blood biochemical indicators.

[0133] After 15 days of feeding: The mice in the control group and the experimental group had normal diet, increased weight, moved freely, had smooth fur, normal urination and defecation, no abnormal secretions, and no abnormal blood biochemical indicators.

[0134] Mice were euthanized at the end of the observation period. No obvious pathological changes were found in the control group and experimental group mice upon dissection.

[0135] This demonstrates that the mycotoxin binder of this application is non-toxic to mice and is safe and reliable.

[0136] Application examples

[0137] Application Example 1

[0138] The compound mycotoxin binder obtained in Example 1 was applied to moldy feed, with a weight ratio of moldy feed to compound mycotoxin binder of 450:1.

[0139] Application Example 2-18

[0140] Unlike Application Example 1, the compound mycotoxin removers in Application Examples 2-18 are derived from Examples 2-18, respectively.

[0141] Application Example 19

[0142] Unlike Application Example 1, the compound mycotoxin remover in Application Example 19 is applied to moldy corn flour.

[0143] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a composite mycotoxin remover, characterized in that, Includes the following steps: S1. Preparation of modified montmorillonite: 1) Dissolve copper sulfate in water to prepare a copper sulfate solution; 2) Add montmorillonite to the copper sulfate solution, and then add a pH adjuster to adjust the pH of the solution to 10; 3) Add formaldehyde solution as a reducing agent and start the chemical deposition reaction at 70-75℃ for 12-14 hours; 4) After the reaction is complete, filter, wash and dry to remove unreacted copper ions and reducing agent residues to obtain modified montmorillonite; S2. Bacillus laterosporus is cultured in a culture medium to obtain Bacillus laterosporus bacterial solution after the culture is completed. Then the obtained bacterial solution is made into dry powder. S3. Mix methyl mercaptoacetate, dimethyl trisulfide, 3-methylbutyrate (S-methyl ester) with water, then add the dry powder to obtain a mixture; S4. Mix the mixture with modified montmorillonite to obtain a composite mycotoxin binder formulation; S5. After drying the compound mycotoxin remover, package it.

2. The method for preparing a composite mycotoxin remover according to claim 1, characterized in that, The concentration of the copper sulfate solution is 16-20 g / L; the weight ratio of copper sulfate to montmorillonite is 1:(5-12); and the weight ratio of formaldehyde to copper sulfate is (3-4):

5.

3. The method for preparing a composite mycotoxin remover according to claim 2, characterized in that, The weight ratio of copper sulfate to montmorillonite is 1:(6-8).

4. The method for preparing a composite mycotoxin remover according to claim 1, characterized in that, The modified montmorillonite is grafted with hexadecyltrimethylammonium bromide, and the grafting method is as follows: a) Dissolve hexadecyltrimethylammonium bromide in water to obtain a hexadecyltrimethylammonium bromide solution, and add methyl p-toluenesulfonate to the hexadecyltrimethylammonium bromide solution to obtain a treatment solution; b) Add the modified montmorillonite to the treatment solution, mix, and then reflux at 70-85℃ for 2-4 hours; c) After the reaction is complete, the slurry is ultrasonically dispersed, the supernatant is removed by centrifugation, the precipitate is collected and washed; the washed precipitate is dried at 80-90℃ to obtain hexadecyltrimethylammonium bromide-modified montmorillonite.

5. The method for preparing a composite mycotoxin remover according to claim 4, characterized in that, The weight ratio of hexadecyltrimethyl bromide to methyl p-toluenesulfonate is 1:(0.1-0.3); the weight ratio of hexadecyltrimethyl bromide to modified montmorillonite is 1:(35-42).

6. The method for preparing a composite mycotoxin binder according to claim 1, characterized in that, In S3, methyl mercaptoacetate, dimethyl trisulfide, 3-methylbutyrate S-methyl ester, 3-mercaptopropyltrimethoxysilane, and water are mixed, and then dry powder is added to obtain a mixture.

7. A composite mycotoxin binder prepared by the method described in any one of claims 1-6, characterized in that, The raw materials include the following parts by weight: 5-15 parts of Bacillus retroflexus, 15-25 parts of methyl mercaptoacetate, 10-20 parts of dimethyl trisulfide, 15-35 parts of 3-methylbutyrate S-methyl ester, 15-30 parts of modified montmorillonite, 20-40 parts of water, and optionally 5-9 parts of 3-mercaptopropyltrimethoxysilane.

8. The composite mycotoxin remover according to claim 7, characterized in that, The weight ratio of methyl mercaptoacetate, dimethyl trisulfide, and 3-methylbutyrate S-methyl ester is 1:(0.6-0.9):(1-1.5).

9. A composite mycotoxin remover according to claim 7, characterized in that, The weight ratio of the methyl mercaptoacetate, dimethyl trisulfide, 3-methylbutyrate S-methyl ester to Bacillus lateralis is (8-12):

1.

10. A composite mycotoxin remover according to claim 7, characterized in that, For use on moldy feed, the weight ratio of moldy feed to compound mycotoxin binder is (400-500):1.